Altered senescence, apoptosis, and DNA damage response in a mutant p53 model of accelerated aging

Abstract

The tumor suppressors p16(INK4a) and p53 have been implicated as contributors to age-associated stem cell decline. Key functions of p53 are the induction of cell cycle arrest, senescence, or apoptosis in response to DNA damage. Here, we examine senescence, apoptosis, and DNA damage responses in a mouse accelerated aging model that exhibits increased p53 activity, the p53(+/m) mouse. Aged tissues of p53(+/m) mice display higher percentages of senescent cells (as determined by senescence-associated beta-galactosidase staining and p16(INK4a) and p21 accumulation) compared to aged tissues from p53(+/+) mice. Surprisingly, despite having enhanced p53 activity, p53(+/m) lymphoid tissues exhibit reduced apoptotic activity in response to ionizing radiation compared to p53(+/+) tissues. Ionizing radiation treatment of p53(+/m) tissues also induces higher and prolonged levels of senescence markers p16(INK4a) and p21, suggesting that in p53(+/m) tissues the p53 stress response is enhanced and is shifted away from apoptosis toward senescence. One potential mechanism for accelerated aging in the p53(+/m) mouse is a failure to remove damaged or dysfunctional cells (including stem and progenitor cells) through apoptosis. The increased accumulation of dysfunctional and senescent cells may contribute to reduced tissue regeneration, tissue atrophy, and some of the accelerated aging phenotypes in p53(+/m) mice.

Fig. 1

p53^+/m MEFs exhibit increased levels of senescence markers compared to p53^+/+ MEFs. Mouse embryonic fibroblasts (MEFs) were passaged and evaluated at passages 2 (P2), 6 (P6) and 10 (P10). (A) SA-β-gal staining of MEFs, as indicated by blue perinuclear staining, reveals increased accumulation of positive staining in p53^+/− MEFs at early passage (P2) and p53^+/m MEFS at late passage (P10) compared to wildtype (p53^+/+) MEFs. (B) Quantitation of the mean percent senescence (±s.e.m.) show that p53^+/m MEFs exhibit significantly higher levels of staining at each passage than p53^+/+ MEFs (*p < 0.02; n = 4 per p53 genotype/passage number). (C) Western blots for cellular senescence initiator p21 and senescence marker p16^INK4a verify increased senescence in p53^+/m MEFs compared to p53^+/+ MEFs with passage.

Fig. 2

p53^+/m tissues exhibit increased levels of senescence markers compared to p53^+/+ tissues. Kidney, liver, and spleen from young 3 month (Y) and old 18–22 month (O) p53^+/−, p53^+/+ and p53^+/m mice were analyzed for markers of cellular senescence. (A) Representative SA-β-gal staining of tissues, as indicated by blue perinuclear staining and arrows. All images are shown at 40× magnification. (B) Quantitation of senescent cells in multiple microscope fields for four mice of each age/genotype group reveals increased accumulation of positive staining in p53^+/m tissues with age as compared to p53^+/+ tissues (*p < 0.05). (C) Western blots for cellular senescence initiator p21 and senescence marker p16^INK4a verify increased senescence in p53^+/m spleen compared to p53^+/+ spleen with age.

Fig. 3

Tissues that exhibit accelerated senescence in p53^+/m mice also show increased mutation frequency with age. Kidney, liver, spleen, and small intestine were examined for mutation frequency in genomic DNA of p53^+/+ and p53^+/m mice at 3, 6, 12, and 24 months of age using the Big Blue Transgenic Mouse system. Wildtype p53 tissue mutation frequencies are represented by light blue lines, p53^+/m mutation frequencies are in dark blue (n ≥ 3 at each data point). Increased mutation frequency is observed in all tissues in both genotypes with age. (A) Kidneys of p53^+/m mice trend towards increased mutations versus p53^+/+ kidneys in the oldest animals (p = 0.051). (B) Livers of 24 month p53^+/m mice exhibit increased mutation frequencies compared to 24 month p53^+/+ livers (p = 0.056). (C) Spleens of p53^+/m mice display increases in mutation frequency with age as compared to p53^+/+ spleens (p = 0.076). (D) In small intestine, a tissue where cellular senescence is neither expected nor reported in the literature, p53^+/+ and p53^+/m mutation frequencies are indistinguishable (p = 0.388).

Fig. 4

Apoptosis is reduced in p53^+/m lymphoid tissues compared to wildtype lymphoid tissues. (A) One week after IR p53^+/+ male spleens exhibit a steep decrease in spleen mass (mg spleen per gram body weight) at IR dosage of 3 Gray (Gy) and 5 Gy, whereas p53^+/m spleens are refractory until the highest levels of IR (n = 3 per data point). p53^+/− spleens exhibit a steady decline in spleen mass with increasing IR dosage. The data are normalized to non-irradiated spleens. (B) Representative TUNEL staining of young and old p53^+/+, p53^+/−, and p53^+/m non-irradiated and 4 h post 5 Gy irradiation spleens. Dark brown-black nuclear staining indicates TUNEL positive, apoptotic cells. All images are shown at 20× magnification. (C) Quantitation of percent average TUNEL positive staining (±s.e.m) in young and old untreated and 5 Gy IR treated spleens from p53^+/+, p53^+/− and p53^+/m animals. In both young and old spleens, p53^+/m tissue sections exhibit significantly lower levels of positive TUNEL staining as compared to p53^+/+ tissue sections (**p < 4.2 × 10⁻⁵ and *p < 0.008, respectively) (n = 4). (D) Western blot showing reduced cleavage of apoptosis-facilitating proteins (caspase 3, caspase 9, and PARP) in young p53^+/m thymus relative to young p53^+/+ thymus, 6 h post 5 Gy IR.

Fig. 5

Increased IR dosage leads to increased and prolonged induction of molecular senescence markers in p53^+/m mice. (A) Six-week-old male p53^+/+ and p53^+/m mice were irradiated with a dosage curve ranging from 0 to 5 Gy IR and spleens were harvested one week post-irradiation. Western blots show little or no increase in p21 or p16^INK4a senescence markers in p53^+/+ spleens, but dramatic increases in both markers in the p53^+/m spleens. (B) Three-month-old p53^+/+, p53^+/−, p53^−/−, and p53^+/m male mice were irradiated with 5 Gy IR and spleens were harvested 6 h post-irradiation. Western blot analysis indicates significant increase in the levels of p16^INK4a in p53^+/m spleens. Levels of p53 protein induced 6 h post IR are also shown. When normalized to loading control, levels of p53 induced in p53^+/m spleens are at least three fold higher than in p53^+/+ spleens. Representative blots are illustrated.

Fig. 6

Models showing relative p53 responses in p53^+/−, p53^+/+, and p53^+/m mice (A) and p53^+/m accelerated organismal and cellular aging resulting from altered stress responses (B). (A) In response to DNA damage and other stresses, p53 induces differential senescence, apoptosis, and damage responses dependent on p53 dosage and activity. Relative responses are indicated by bar heights for each effect for p53^+/−, p53^+/+, and p53^+/m mice. p53^+/m mice exhibit enhanced senescence and DNA damage responses and reduced apoptosis relative to p53^+/+ mice. (B) Enhanced damage responses in p53^+/m tissues are associated with inadequate apoptotic removal of damaged cells and retention of senescent cells in p53^+/m tissues and may contribute to the accelerated aging phenotypes observed in p53^+/m mice. As p53^+/+ mice age, damaged cells are efficiently removed by p53-mediated apoptosis and replaced by functional adult tissue stem cells (top part of panel). Senescent cells accumulate at a modest rate and aging phenotypes occur late in life. In contrast, p53^+/m tissues exhibit enhanced anti-proliferative damage responses and are defective in apoptotic elimination of damaged or dysfunctional cells and these accumulate with age, perhaps as senescent cells. Such cells may include stem and progenitor cells that more rapidly lose the ability to replenish cells in the tissues as the organism ages. The result is tissue atrophy and some of the accelerated aging phenotypes observed in the p53^+/m mouse.